Fusion of male and female gametes to form a zygote during fertilization is the defining moment in the life of a eukaryote. Although understanding gamete fusion is critical for reproductive health, we do not yet know for even a single organism the neither molecules nor molecular steps required for fusion of gamete membranes. My laboratory uses the biflagellated, unicellular green alga Chlamydomonas reinhardtii as a model system to study fertilization. In the first phase of fertilization, adhesion between the flagella of plus and minus gametes brings them together and also activates both to expose cell membrane sites specialized for fusion. Next, the fusogenic plasma membranes come into intimate contact and immediately fuse. For the first time in any organism, we have now shown that attachment of fusogenic membranes and merger of the two membranes are genetically distinguishable and are carried out by at least two different integral membrane proteins. Pre- fusion attachment between gamete membranes is governed by a species-specific plus gamete-specific protein FUS1, and subsequent membrane merger depends on a broadly conserved, minus gamete-specific protein, HAP2. HAP2 family members are present in sponges; cnidarians; several insects; higher plants; and many devastating pathogenic protists, including Plasmodium. Furthermore, we have also uncovered a molecular mechanism for a membrane block to polyspermy, demonstrating that both FUS1 and HAP2 undergo rapid, fusion-dependent proteolysis during a Chlamydomonas membrane block to polygamy. This Chlamydomonas system is poised to allow us to dissect the molecular mechanisms of gamete fusion using knowledge and approaches not yet available for other systems. We have well-established bioassays to detect and quantify each step in gamete interactions; we have sterile mutants blocked at several steps in fertilization; the organism is easily amenable to genetic and molecular biological manipulations; and, we can prepare quantities of protein sufficient for biochemistry and structural studies. Our discovery of HAP2 already has had an unexpected impact in malaria fertilization research. With our collaborators we showed that HAP2 is essential for Plasmodium gamete fusion and mosquito transmission of malaria, and therefore a new prime target for a malaria transmission-blocking vaccine. Understanding, for at least one organism, the molecular events that occur during the gamete membrane fusion reaction will have a large impact on the field of reproductive biology. Such knowledge will establish a framework for dissecting fundamental principles of gamete fusion in other organisms and for development of contraceptives and for treating infertility. The objective of the research strategy presented here is to test the model that HAP2 functions as a fusion protein during the membrane fusion reaction. We will identify proteins that interact with HAP2, we will study the functional domains of HAP2, and we will identify new proteins that function during membrane fusion.